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Exploring the microbial diversity associated with
coral reef environments.                      -Zeenat Tinwala
                                                      1072448
                                               April 10th 2013
What is a coral reef?
 Most biologically diverse ecosystems
 Not only of hard and soft corals
 Fluctuations - alter both the diversity and abundances of others
 Water filtration and fish reproduction to shore line protection and erosion
 prevention.




                                                                                2
Locations
 Great Barrier Reef—largest-off Queensland, Australia
 Mesoamerican Barrier Reef System—second largest
 New Caledonia Barrier Reef—second longest double barrier reef




             http://www.nasa.gov/vision/earth/lookingatearth/coralreef_image.html   3
Biodiversity
 World's most productive ecosystems (Barnes et al., 1991)
 Fish, seabirds, sponges, cnidarians, worms, crustaceans, mollusks,
 echinoderms, sea squirts, sea turtles and sea snakes
 Mutually beneficial relationship with a microscopic unicellular
 Reefs are chronically at risk of algal encroachment. (Glynn, 1990)




                                                                       4
Microbial Diversity
 Coral-algal symbiosis but little is known about other microbes
 Microbial assemblage/ Coral holobiont
 The microbial community fluctuate along seasonal and biogeographic ranges
 Roles that microorganisms play in coral physiology and immune function -
 unknown
 Beneficial microbial assemblages - Antibiotics
 Detecting when and why shifts occur in healthy coral-microbial assemblages is
 to a large extent unknown.


                                                                    (Morrow, et al., 2012)

                                                                                             5
(Ainsworth, Thurber, & Gates, 2010)


                     6
Targeted goals of research
 Understand the microbial communities
 Change through time – Key to understand health of coral reefs
 Same species - different locations - distinct microbial communities
 Whether macroalgal extracts cause a detectable stress response in coral tissues
 and/or a shift in coral-associated bacterial assemblages.




                                                         (Morrow, Ritson-Williams, Ross, Liles, & Paul, 2012)


                                                                                                                7
Methods used
 PCR amplification of 16S ribosomal gene
 DGGE analysis
 T-RFLP Terminal Restriction Fragment Length Polymorphism
 R-Project
 SSCP Single Stranded Conformation Polymorphism
 nMDS – Non-metric multidimensional scaling plot
 Sequence Analysis
  CHECK_CHIMERA
  CLUSTALX
  PAUP



                                                             8
(Ainsworth, Thurber, & Gates, 2010)


                     9
Characterization of microbial
community function
 Phylogenetic markers
 Example: cyanobacteria Prochlorococcus strains- almost identical 16S
 sequences (97% similarity), but differ physiologically
 Vibrio splendidus - 70% of the 206 strains that make up a single Vibrio
 splendidus ribotype cluster have differences in genome size even though the 16S
 genes of these strains are <1% divergent




                                                                     (Rocap, G.et al. 2003)

                                                                                     10
Factors influencing Coral-Microbe
interaction




                                    (Sokolow, 2009)


                                                11
Exploring a few locations
 Caribbean and Indo-Pacific Reefs
 Great Barrier Reef
 South China Sea
 Ningaloo Reef




                                     © Gary Bell/Oceanwidelmages.com
                                                                       12
Caribbean & Indo-Pacific Reefs
 Dominant component of Black Band Disease (BBD)- cyanobacteria –
 Phormidium corallyticum
 These cyanobacteria belong to at least 3 different taxa, the corals in each case
 exhibit similar signs and patterns of BBD mat development.
 Environmental factors that lead to the development of BBD- Unknown
 The genetic diversity among the cyanobacteria that are part of the BBD mat -
 help explain why BBD has been observed under many different environmental
 conditions.
 Macroalgae both stimulated and inhibited coral reef-associated bacterial
 cultures.
                                                   (Frias-Lopez, Bonheyo, Jin, & Fouke, 2003; Morrow KM, Paul VJ, Liles MR, 2011)


                                                                                                                    13
Great Barrier Reef
 3 species of Acropora were compared at two locations (Magnetic Island and Orpheus
Island)
 Sequences from duplicate clone libraries of each species were consistent
  Gammaproteobacteria - Dominant
  Alphaproteobacteria
  Deltaproteobacteria

 Oculina patagonicain - infection with the bacterium Vibrio shiloi - Seasonal bleaching
Vibrio coralliilyticus - aetiological agent of bleaching and tissue lysis in Pocillopora
 damicornis
 Vibro shiloi and Vibrio coralliilyticus have been confirmed as coral bleaching pathogens

                                                  (Banin et al., 2000; Ben-Haim and Rosenberg, 2002; Rosenberg and Ben-Haim, 2002)


                                                                                                                         14
South China Sea
 5 species of gorgonian corals
 collected in shallow waters.
 123 actinobacterial isolates were
 recovered using 10 different isolation
 media, and assigned to 11 genera
Streptomyces and Micromonospora -
 dominant genera
 Actinobacteria could protect their
 hosts against marine pathogens.


                                          (Zhang et al., 2013)

                                              15
Ningaloo Reef
Effect of heat stress on coral-associated bacterial, Acropora tenuis, hosting
 different Symbiodinium types
 Increase in the number of retrieved Vibrio affiliated sequences, these
 sequences affiliated most closely with the coral pathogen, Vibrio coralliilyticus
 Thermal stress - shifts in coral-associated bacterial communities - deteriorating
 coral health.
 Bacterial 16S rRNA clone were highly diverse, though dominated by a- and c-
 Proteobacteria
 Responses of coral-associated bacterial communities exposed to heat stress can
 differ for juvenile corals hosting different Symbiodinium partners.
                                                                  Littman, Bourne, & Willis, 2010)

                                                                                             16
Future directions
 Understand the role of specific microbial species in biogeochemical cycles.
Metagenomic and qPCR-based approaches combined with culture-independent
 technologies such as single-cell genomics.
High-resolution fluorescent in situ hybridization and scanning electron
 microscopy - where and when microbial species are established in host corals.
 Phase-shifts to alternative dominants that may have potent biochemical
 defence mechanisms - what effect this will have on overall reef health and
 physiology.



                                                      (Morrow, Ritson-Williams, Ross, Liles, & Paul, 2012)

                                                                                                   17
Refernces
Ainsworth, T. D., Thurber, R. V., & Gates, R. D. (2010). The future of coral reefs: a microbial
 perspective. Trends Ecol Evol, 25(4), 233-240. doi: 10.1016/j.tree.2009.11.001
Frias-Lopez, J., Bonheyo, G. T., Jin, Q., & Fouke, B. W. (2003). Cyanobacteria associated with coral
 black band disease in Caribbean and Indo-Pacific Reefs. Appl Environ Microbiol, 69(4), 2409-2413.
Littman, R. A., Bourne, D. G., & Willis, B. L. (2010). Responses of coral-associated bacterial
 communities to heat stress differ with Symbiodinium type on the same coral host. Mol Ecol, 19(9),
 1978-1990. doi: 10.1111/j.1365-294X.2010.04620.x
Morrow, K. M., Ritson-Williams, R., Ross, C., Liles, M. R., & Paul, V. J. (2012). Macroalgal extracts
 induce bacterial assemblage shifts and sublethal tissue stress in Caribbean corals. PLoS One, 7(9),
 e44859. doi: 10.1371/journal.pone.0044859
Sokolow, S. (2009). Effects of a changing climate on the dynamics of coral infectious disease: a review
 of the evidence. Dis Aquat Organ, 87(1-2), 5-18. doi: 10.3354/dao02099
Zhang, X. Y., He, F., Wang, G. H., Bao, J., Xu, X. Y., & Qi, S. H. (2013). Diversity and antibacterial activity
 of culturable actinobacteria isolated from five species of the South China Sea gorgonian corals. World
 J Microbiol Biotechnol. doi: 10.1007/s11274-013-1279-3


                                                                                                               18
Banin, E., Israely, T., Kusmaro, A., Loya, Y., Orr, E., and Rosenberg, E. (2000) Penetration of the
 coral-bleaching bacterium Vibrio shiloiinto Oculina patgonica. Appl Envi-ron Microbiol 66: 3031–
 3036.
Banin, E., Israely, T., Fine, M., Loya, Y., and Rosenberg, E. (2001a) Role of endosymbiotic
 zooxanthellae and coral mucus in the adhesion of the coral-bleaching pathogen Vibrio shiloito its
 host. FEMS Microbiol Lett 199: 33–37.
Ben-Haim, Y., and Rosenberg, E. (2002) A novel Vibriosp. pathogen of the coral Pocillopora
 damicornis. Mar Biol 141:47–55.
Barnes, R.S.K., and Mann, K.H. (1991). Fundamentals of Aquatic Ecology. Blackwell Publishing.
 pp. 217–227. ISBN 0-632-02983-8. Retrieved November 26, 2008.
Glynn, P.W. (1990). In Dubinsky, Z. Ecosystems of the World v. 25-Coral Reefs. New York, NY:
 Elsevier Science.
Rocap, G.et al. (2003) Genome divergence in two Prochlorococcus ecotypes reflects oceanic
 niche differentiation.Nature424, 1042–1047
Morrow KM, Paul VJ, Liles MR, Chadwick NE (2011) Allelochemicals produced by Caribbean
 macroalgae and cyanobacteria have species-specific effects on reef coral microorganisms. Coral
 Reefs 30: 309–320.



                                                                                                  19
Thank you!!

              20
Questions?

             21

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Exploring the microbial diversity associated with coral reef environments.

  • 1. Exploring the microbial diversity associated with coral reef environments. -Zeenat Tinwala 1072448 April 10th 2013
  • 2. What is a coral reef?  Most biologically diverse ecosystems  Not only of hard and soft corals  Fluctuations - alter both the diversity and abundances of others  Water filtration and fish reproduction to shore line protection and erosion prevention. 2
  • 3. Locations  Great Barrier Reef—largest-off Queensland, Australia  Mesoamerican Barrier Reef System—second largest  New Caledonia Barrier Reef—second longest double barrier reef http://www.nasa.gov/vision/earth/lookingatearth/coralreef_image.html 3
  • 4. Biodiversity  World's most productive ecosystems (Barnes et al., 1991)  Fish, seabirds, sponges, cnidarians, worms, crustaceans, mollusks, echinoderms, sea squirts, sea turtles and sea snakes  Mutually beneficial relationship with a microscopic unicellular  Reefs are chronically at risk of algal encroachment. (Glynn, 1990) 4
  • 5. Microbial Diversity  Coral-algal symbiosis but little is known about other microbes  Microbial assemblage/ Coral holobiont  The microbial community fluctuate along seasonal and biogeographic ranges  Roles that microorganisms play in coral physiology and immune function - unknown  Beneficial microbial assemblages - Antibiotics  Detecting when and why shifts occur in healthy coral-microbial assemblages is to a large extent unknown. (Morrow, et al., 2012) 5
  • 6. (Ainsworth, Thurber, & Gates, 2010) 6
  • 7. Targeted goals of research  Understand the microbial communities  Change through time – Key to understand health of coral reefs  Same species - different locations - distinct microbial communities  Whether macroalgal extracts cause a detectable stress response in coral tissues and/or a shift in coral-associated bacterial assemblages. (Morrow, Ritson-Williams, Ross, Liles, & Paul, 2012) 7
  • 8. Methods used  PCR amplification of 16S ribosomal gene  DGGE analysis  T-RFLP Terminal Restriction Fragment Length Polymorphism  R-Project  SSCP Single Stranded Conformation Polymorphism  nMDS – Non-metric multidimensional scaling plot  Sequence Analysis  CHECK_CHIMERA  CLUSTALX  PAUP 8
  • 9. (Ainsworth, Thurber, & Gates, 2010) 9
  • 10. Characterization of microbial community function  Phylogenetic markers  Example: cyanobacteria Prochlorococcus strains- almost identical 16S sequences (97% similarity), but differ physiologically  Vibrio splendidus - 70% of the 206 strains that make up a single Vibrio splendidus ribotype cluster have differences in genome size even though the 16S genes of these strains are <1% divergent (Rocap, G.et al. 2003) 10
  • 12. Exploring a few locations  Caribbean and Indo-Pacific Reefs  Great Barrier Reef  South China Sea  Ningaloo Reef © Gary Bell/Oceanwidelmages.com 12
  • 13. Caribbean & Indo-Pacific Reefs  Dominant component of Black Band Disease (BBD)- cyanobacteria – Phormidium corallyticum  These cyanobacteria belong to at least 3 different taxa, the corals in each case exhibit similar signs and patterns of BBD mat development.  Environmental factors that lead to the development of BBD- Unknown  The genetic diversity among the cyanobacteria that are part of the BBD mat - help explain why BBD has been observed under many different environmental conditions.  Macroalgae both stimulated and inhibited coral reef-associated bacterial cultures. (Frias-Lopez, Bonheyo, Jin, & Fouke, 2003; Morrow KM, Paul VJ, Liles MR, 2011) 13
  • 14. Great Barrier Reef  3 species of Acropora were compared at two locations (Magnetic Island and Orpheus Island)  Sequences from duplicate clone libraries of each species were consistent  Gammaproteobacteria - Dominant  Alphaproteobacteria  Deltaproteobacteria  Oculina patagonicain - infection with the bacterium Vibrio shiloi - Seasonal bleaching Vibrio coralliilyticus - aetiological agent of bleaching and tissue lysis in Pocillopora damicornis  Vibro shiloi and Vibrio coralliilyticus have been confirmed as coral bleaching pathogens (Banin et al., 2000; Ben-Haim and Rosenberg, 2002; Rosenberg and Ben-Haim, 2002) 14
  • 15. South China Sea  5 species of gorgonian corals collected in shallow waters.  123 actinobacterial isolates were recovered using 10 different isolation media, and assigned to 11 genera Streptomyces and Micromonospora - dominant genera  Actinobacteria could protect their hosts against marine pathogens. (Zhang et al., 2013) 15
  • 16. Ningaloo Reef Effect of heat stress on coral-associated bacterial, Acropora tenuis, hosting different Symbiodinium types  Increase in the number of retrieved Vibrio affiliated sequences, these sequences affiliated most closely with the coral pathogen, Vibrio coralliilyticus  Thermal stress - shifts in coral-associated bacterial communities - deteriorating coral health.  Bacterial 16S rRNA clone were highly diverse, though dominated by a- and c- Proteobacteria  Responses of coral-associated bacterial communities exposed to heat stress can differ for juvenile corals hosting different Symbiodinium partners. Littman, Bourne, & Willis, 2010) 16
  • 17. Future directions  Understand the role of specific microbial species in biogeochemical cycles. Metagenomic and qPCR-based approaches combined with culture-independent technologies such as single-cell genomics. High-resolution fluorescent in situ hybridization and scanning electron microscopy - where and when microbial species are established in host corals.  Phase-shifts to alternative dominants that may have potent biochemical defence mechanisms - what effect this will have on overall reef health and physiology. (Morrow, Ritson-Williams, Ross, Liles, & Paul, 2012) 17
  • 18. Refernces Ainsworth, T. D., Thurber, R. V., & Gates, R. D. (2010). The future of coral reefs: a microbial perspective. Trends Ecol Evol, 25(4), 233-240. doi: 10.1016/j.tree.2009.11.001 Frias-Lopez, J., Bonheyo, G. T., Jin, Q., & Fouke, B. W. (2003). Cyanobacteria associated with coral black band disease in Caribbean and Indo-Pacific Reefs. Appl Environ Microbiol, 69(4), 2409-2413. Littman, R. A., Bourne, D. G., & Willis, B. L. (2010). Responses of coral-associated bacterial communities to heat stress differ with Symbiodinium type on the same coral host. Mol Ecol, 19(9), 1978-1990. doi: 10.1111/j.1365-294X.2010.04620.x Morrow, K. M., Ritson-Williams, R., Ross, C., Liles, M. R., & Paul, V. J. (2012). Macroalgal extracts induce bacterial assemblage shifts and sublethal tissue stress in Caribbean corals. PLoS One, 7(9), e44859. doi: 10.1371/journal.pone.0044859 Sokolow, S. (2009). Effects of a changing climate on the dynamics of coral infectious disease: a review of the evidence. Dis Aquat Organ, 87(1-2), 5-18. doi: 10.3354/dao02099 Zhang, X. Y., He, F., Wang, G. H., Bao, J., Xu, X. Y., & Qi, S. H. (2013). Diversity and antibacterial activity of culturable actinobacteria isolated from five species of the South China Sea gorgonian corals. World J Microbiol Biotechnol. doi: 10.1007/s11274-013-1279-3 18
  • 19. Banin, E., Israely, T., Kusmaro, A., Loya, Y., Orr, E., and Rosenberg, E. (2000) Penetration of the coral-bleaching bacterium Vibrio shiloiinto Oculina patgonica. Appl Envi-ron Microbiol 66: 3031– 3036. Banin, E., Israely, T., Fine, M., Loya, Y., and Rosenberg, E. (2001a) Role of endosymbiotic zooxanthellae and coral mucus in the adhesion of the coral-bleaching pathogen Vibrio shiloito its host. FEMS Microbiol Lett 199: 33–37. Ben-Haim, Y., and Rosenberg, E. (2002) A novel Vibriosp. pathogen of the coral Pocillopora damicornis. Mar Biol 141:47–55. Barnes, R.S.K., and Mann, K.H. (1991). Fundamentals of Aquatic Ecology. Blackwell Publishing. pp. 217–227. ISBN 0-632-02983-8. Retrieved November 26, 2008. Glynn, P.W. (1990). In Dubinsky, Z. Ecosystems of the World v. 25-Coral Reefs. New York, NY: Elsevier Science. Rocap, G.et al. (2003) Genome divergence in two Prochlorococcus ecotypes reflects oceanic niche differentiation.Nature424, 1042–1047 Morrow KM, Paul VJ, Liles MR, Chadwick NE (2011) Allelochemicals produced by Caribbean macroalgae and cyanobacteria have species-specific effects on reef coral microorganisms. Coral Reefs 30: 309–320. 19

Hinweis der Redaktion

  1. http://reefrelief.org/learn/coral-reef-ecosystem/ Not only of hard and soft corals, but also sponges, crustaceans, mollusks, fish, sea turtles, sharks, dolphins and much more
  2. The Andros, Bahamas BarrierThe Red Sea—includes 6000-year-old fringing reefs The Florida Reef Tract—largest continental US reefPulley Ridge—deepest photosynthetic coral reef, FloridaNumerous reefs scattered over the MaldivesThe Philippines coral reef area
  3. Ecosystem - providing complex and varied marine habitatscnidarians (which includes some types of corals and jellyfish)crustaceans (including shrimp, cleaner shrimp, spiny lobsters and crabs), algae called zooxanthellae that lives within the cells of the coral&apos;s gastrodermis.
  4. Microbial assemblage - referred to as the coral holobiont, encompasses a wide range of taxa, including numerous microbial eukaryotes, archaea, bacteria, and viruses. Some microbes can provide protection against bacterialor fungal infection [8] by the production of antibiotics or by filling a niche that otherwise would be open to infection by opportunistic pathogens [9].
  5. The complexity of the coral reef structure results in many microhabitats. The host structure provides various microhabitats for microbial colonization within thecoral colony, coral polyps and coral tissues. Each microbial compartment on the reef is influenced by physical and biological environmental conditions that vary in time andspace. Environmental variability through the water column (dark blue arrow) is related to reef depth and reefal position. Biological variability along the branch axis (brownarrow) is related to environmental (light and water flow) and biological factors (colony openness, endosymbioticdinoflagellate density, respiration and photosynthesis).Variability along the branch apical to the basel axes (green arrow) is related to the variable 3-dimension structure of host, polvp density, and nicheenvironments formicrobial colonization and biofilm formation.
  6. DGGE : Denaturing gradient gel electrophoresis
  7. Phylogenetic markers, such as the small subunit of the ribosome, are useful for characterizing the diversity of the microbial community but reveal little about the metabolic capabilities of organisms
  8. The genetic diversity among the cyanobacteria that are part of the BBD mat is a new discovery that may help explain why BBD has been observed under many different environmental conditionsmacroalgae and the compounds they produce can alter microbial assemblages associated with reef-building corals
  9. (Acroporamillepora, Acroporatenuis and Acroporavalida) the Alphaproteobacteria, which comprised between 10% and 33% of retrieved sequences, while a small proportion of clones affiliated within the Deltaproteobacteria class, constituting between 1% and 12% of the libraries bacterium Vibrio shiloi,in which a number of virulence factors appear to be produced only at elevated temperatures, associated with the onset of bleachingPocilloporadamicornis,amajor reef-building coral found throughout the Great Barrier Reef (GBR)Vibrio coralliilyticus, is the aetiological agent of bleaching and tissue lysis in the coral Pocilloporadamicornis
  10. Relative composition ofthe 123 actinobacterial strainsisolated from the five species ofgorgonian corals from the SouthChina Sea5 species - (Echinogorgiaaurantiaca, Melitodessquamata, Muricellaflexuosa,Subergorgiasuberosa, and Verrucellaumbraculum)
  11. The lower resilience ofA. tenuisto thermal stress when harbouringSymbiodiniumD highlights the importanceof inter-kingdom interactions among the coral host, dinoflagellateendosymbiont andbacterial associates for coral health and resilience.For example, under heat stress we foundminimal changes in bacterial communities associatedwith juveniles of the coral,Acroporatenuis,when ithostedSymbiodiniumC1. In contrast, distinct shifts inbacterial communities on A. tenuis juveniles hostingSymbiodinium D were observed when exposed to thesame elevated temperature treatment.Bacterial 16S rRNA clone libraries derived from both C1- and D-juveniles were highly diverse, though dominated by a- and c-Proteobacteria affiliated sequences